1. Field of the Invention
The invention relates to a method for producing large quantities of human parathyroid hormone 1-84 (hPTH 1-84) or variants thereof in bacteria using recombinant DNA techniques.
2. Description of the Background Art
Parathyroid hormone (PTH) is an 84-amino acid residue peptide and one of the main regulators of calcium homeostasis in the mammalian organism. PTH acts primarily on the kidney, stimulating calcium resorption, and on bone cells, stimulating calcium mobilization. PTH also leads to enhancement of the bone remodeling process (sec Potts et al. Adv. Protein Chem. 32:323-395 (1982)). PTH may have promising effects in the therapy or prevention of osteoporosis.
Variants or analogs of PTH can bind to PTH receptors and block the effects of either PTH, or of the recently discovered PTH-Rotated Peptide, which is produced by a number of tumors such as renal cell carcinoma and carcinomas of the lung and breast, and is now thought to be responsible for hypercalcemia of malignancy (Broadus, A. et al., New. Eng. J. Med. 319:356-363 (1988)).
For the foregoing reasons, a source of large quantities of easily purified human PTH has been sought for a number of years.
Several reports have described direct expression of PTH, in E. coli (Breyel et al., 3rd Europ. Cong. Biotech. 3:363-369 (1984); Born, W. et al., Endocrinol. 123:1848-1853 (1988); Morelle, et al., Biochim. Biophys. Acta 950 459-462 (1988); Rabbani et al. J. Biol. Chem. 263:1307-1313 (1988)). The attempts of Breyel et al. and Morelle et al. (supra) to express human PTH yielded only very limited amounts of hormone ( less than 500 xcexcg/liter) due to RNA and protein instability. Rabbani et al. (supra) achieved yields of 200 xcexcg liter PTH, which was contaminated with peptides variously truncated at the N-terminus. Once the pure intact PTH(1-84) was purified by an intricate method, the final yield was only 10 xcexcg/liter culture. Born et al. (supra), using a multicopy plasmid of human preproPTH cDNA, achieved expression of truncated hPTH, largely the PTH(3-84) and PTH(8-84), which were virtually devoid of PTH bioactivity.
Thus, the need was recognized for an expression vector which would lead to production of higher quantities of stable PTH, that could be easily purified, and was devoid of inactive forms/analogs of PTH. One approach to the solution of this problem was suggested by work describing expression of other peptides in bacterial hosts (Itakura et al., Science 198:1036-1063 (1977); Riggs, U.S. Pat. No. 4,366,246, Method for Microbial Polypeptide Expression, (Dec. 28, 1982); Ikehara et el., Proc. Natl. Acad. Sci. USA 81:3956-5960 (1984)).
Itakura et al. (1977, supra) and Riggs (1984, supra) taught methods for bacterial expression of a foreign protein, specifically the production of a fusion protein between a desired heterologous protein (human somatostatin) and a large bacterial protein (betagalactosidase). The fusion protein served the function of preventing intracellular degradation of the foreign protein. Furthermore, overexpressed fusion proteins, especially with xcex2-galactosidase, are frequently deposited as inclusion bodies in the bacterial cells, and are therefore easier to isolate and purify (Marston, Biochem. J. 240:1-12 (1986)). The somatostatin gene was inserted in phase at the C-terminus of the xcex2-galactosidase gene, and the presence of a methionine at the N-terminus of the somatostatin sequence was put in to allow later cleavage of the fusion protein by cyanogen bromide. An inducible bacterial promoter, the lac promoter was inserted to stimulate transcription of the fusion protein mRNA. Amino acid codons known to be favored in E. coli for the production of non-bacterial proteins (e.g. MS2 phage) were utilized. The fusion protein was solubilized with 70% formic acid, 6M guanidinium HCl, 8M urea, or 2% SDS, and the desired product, somatostatin, was subsequently enriched using CNBr cleavage, alcohol extraction, and chromatography. Suggestion was made to alternate cleavage methods based on the concept of selective cleavage sites: Just as methionine could be inserted at the desired junction to serve as a target site for cyanogen bromide, so too could specific amino acid sites be introduced which could be attacked by proteolytic enzymes with amino acid specificity.
Ikehara et al. (1984, supra) described a plasmid containing the E. coli trp promoter which promotes the expression of a synthetic human gene for growth hormone (hGH), leading to enhanced synthesis of the 191 amino acid (hGH), peptide. The synthetic gene utilized amino acid colons most frequent in E coli. 
Nagai et al. (Nature 309:810-812 (1984) pointed out the limitations on high level expression of eukaryotic genes in E. coli, even when appropriate promoters and ribosomal binding sequences have been introduced, and disclosed the production of the desired gene product in a fusion protein as a useful method to increase efficiency easy separation of the desired peptide. To achieve this goal, Nagai et al. introduced a sequence of 4 amino acids which serve as a cleavage site for the blood coagulation factor Xa, and achieved expression of the human beta globin gene fused to part of the phage lambda cII gene. Cleavage by Factor Xa eliminated the extra N-terminal methionine encoded by the initiation colon which is present in most eukaryotic proteins expressed in E. coli by conventional methods.
Other documents describe the design and use of plasmids coding for fusion proteins between a desired heterologous protein and a second protein, some of which include cleavage sites recognized by Factor Xa or thrombin, for enhanced expression in bacteria and greater ease of purification of the desired protein (Germino et al., Proc. Natl. Acad. Sci. USA 81:692-4696 (1984); Scholtissek et al., Gene 62:55-64 (1988);Smith et al., Gene 67:31-40 (1988); Knott et al., Eur. J. Biochem. 174:403-410 (1988); and Dykes et al., Eur. J. Biochem. 174:411-416 (1988)).
Wingender et al., J. Biol. Chem. 264:4367-4373 (1989)) attempted to apply the above-mentioned approaches in order to achieve enhanced expression of hPTH in E. coli. Attempts to introduce the factor Xa recognition site into an expression plasmid, upstream from the hPTH site, as described by Nagai et al. (1985, supra) and to achieve isolation of hPTH from the fusion protein using proteolytic cleavage with factor Xa were unsuccessful, even when highly purified fusion protein and proteinase were used.
The availability of hPTH and analogs which are selectively antagonists and can block the untoward effects mediated by agonists acting at PTH receptors in certain pathologic states would be of great clinical value. For this purpose, a prokaryotic expression system would be of great utility. Such a system would provide not only sufficient material for clinical and research use, but also would allow application of protein engineering methods to investigate structure-function relationships and to design hormone variants to perform specific tasks (e.g. calcium mobilization vs. bone reconstruction, agonistic vs. antagonistic effects).
This invention pertains to a recombinant DNA molecule coding for human parathyroid hormone capable of being expressed efficiently in bacteria. The DNA molecule comprises a control region for expression of a genetic sequence in a host, said region being in operable linkage with a leader sequence coding for a polypeptide sequence, said leader sequence being no more than about 600 nucleotides in length, and being operably linked to a nucleotide sequence coding for a selective enzymatic cleavage site, said nucleotide sequence being linked to and preceding said genetic sequence to be expressed.
The invention is additionally directed to a fusion protein comprising a leader polypeptide, a selected enzymatic cleavage site, and hPTH(1-84) or its variants.
The invention also includes a method for producing large quantities of human parathyroid hormone 1-84 (hPTH 1-84), or variants thereof, in bacteria using the said recombinant DNA molecule.
One of the major advantages of this invention is the ability to produce high concentrations of purified hPTH 1-84, which is a 50-100 fold improvement over currently attainable levels.
The invention enables production of a stable form of hPTH 1-84 resistant to bacterial intracellular degradation, which lacks the added N-terminal methionine which, if present, would render the hormone preparation less active and more immunogenic to a human host.
The invention also permits one to tailor the plasmid construct for production of hPTH analogues with variant N-termini which could serve as useful parathyroid hormone agonists or antagonists.